Purification of .BETA.-lactamase from Streptomyces cellulosae by affinity chromatography on blue sepharose.

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Abstract
A .beta.-lactamase from culture supernatant of S. cellulosae was purified about 1450-fold to apparent homogeneity in polyacrylamide gel electrophoresis and isoelectric focusing on polyacrylamide gel sheet. The methods used were ammonium sulfate precipitation, CM-52 cellulose ion-exchange chromatography and affinity chromatography on Blue Sepharose CL-6B. The MW was determined to be approximately 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was in good agreement with the previous value determined by gel filtration on Sephadex G-75. The isoelectric point was pH 9.5. The enzyme behaved primarily as penicillinase and apparent Km value for benzylpenicillin was 500 .mu.M. The .beta.-lactase of S. cellulosae interacted strongly with blue dextran and NADP+-agarose but not with Sepharose. In addition, the presence of NADP+ but not NAD+ and ATP diminished sharply the intrinsic fluorescence intensity of the enzyme and the apparent association constant was calculated to be 1.4 .times. 103 M-1. The .beta.-lactamase decreases its enzymatic activity against benzylpenicillin in the presence of NADP+. From these results, it is suggested that this .beta.-lactamase has a dinucleotide binding fold.